Project Summary. ?Nuclear magnetic resonance (NMR) is amongst the most powerful analytical techniques ever invented, as recognized by 6 Nobel Prizes for methods development alone. Nonetheless, NMR is notoriously plagued by poor sensitivity and spatial resolution. State-of-the-art NMR spectrometers typically feature detection thresholds of ~100 nanograms for L sample volumes. This places NMR sensitivity many orders of magnitude behind other analytical chemistry techniques such as mass spectrometry, Raman spectroscopy, and fluorescence labeling. Improvements in NMR often focus on using larger magnets, but progress has largely plateaued; over the last 25 years, the fundamental signal strength has only increased by a factor of ~2. We seek to fundamentally change the NMR hardware by using diamond films doped with Nitrogen-Vacancy (NV) centers to detect nuclear magnetization ?non-inductively ?via pulsed optically detected magnetic resonance methods. ?Recently, we performed diamond NMR spectroscopy of 1 pL-volume solutions and demonstrated ~100x improvement in concentration sensitivity over previous NV and picoliter NMR studies. Here we propose to improve the sensitivity (to < 1 pg) and spectral resolution (to <1 ppm) to realize an NMR spectrometer capable of quantitatively differentiating metabolites in sub-nL mixtures at physiologically-relevant concentrations. ?Such a prototype could have a profound impact on metabolomics and pharmacodynamics research, combining mass-spectrometry-level sensitivity with NMR-level accuracy in unrefined samples containing numerous metabolites at sub-mM concentrations.? ?Specifically, we improve upon existing analytical methods by offering: 1. Greater performance?. Our diamond NMR spectrometer will feature several orders of magnitude greater mass sensitivity (picograms instead of nanograms) compared to current NMR spectrometers. The mass sensitivity approaches that of mass spectrometry but retains benefits of NMR such as non-destructive, absolute quantitation and structural identification. 2. Compatibility with hyphenated separation assays?. Our spectrometer is compact and easily integrated into microfluidic chips for online chromatography-based assays (HPLC) for sample-limited analyses (metabolomics, pharmacodynamics, natural products)?. 3. Lower cost. Our spectrometer?s sub-nL detection volume leads to reduced engineering costs (for, e.g., magnet stability), providing greater affordability than current NMR spectrometers.